The present invention relates generally to the field of spectrophotometric analysis of blood serum samples, and more particularly, to the field of kinetic and end point spectrophotometric determinations of triglycerides, cholesterol, calcium, magnesium, glucose, urea, albumin, total proteins, gamma-glutamyl transferase, and uric acid.
It has long been known that blood serum may be spectrophotometrically analyzed by combining a serum sample with one or more selected reagents which will colorimetrically combine with a selected component within that sample. Upon a subsequent spectrophotometric analysis of that sample, the concentration of that component within that sample may be determined.
More recently, it has been suggested that multiple component end point determinations may be made within a single reaction medium. For example, in Chem Abstracts 88 (1978) 83042, (referencing Chemical Analysis (Warsaw) 1977, 22(1), 27-35, entitled "Optimization of the Spectrophotometric Multi-Component Analysis. The Use of a Complex Colorimetric Reagent."), it is suggested that a reagent be composed of two or more compounds may be reacted with two or more of the components of a test solution to give two colored products. The analysis procedure may be simplified if the reagent also includes all auxiliary compounds used in analysis, as for example, buffers, masking agents, etc. The optimization scheme disclosed in this abstract includes the selection of preferred conditions of analysis; that is, preferred colorimetric reagent compositions and preferred wavelengths suited for use during a certain multi-component spectrophotometric analysis. In particular, this abstract discloses a reagent composed of murexide, calmagite, and other materials for the detection of both calcium and magnesium in a given serum sample.
It has also been proposed to make kinetic determinations of the enzymatic activities exhibited by a plurality of enzymes contained in a single aqueous reaction medium. In accordance with this proposed method, known quantities of substrates, one of which is "consumed" by each of the enzymes to be determined, and any reagents required for the measurement of substrate or reaction product concentrations at preselected wavelengths may be added to the reaction medium and as employed permit enzymatic reactions to proceed simultaneously under the same reaction conditions. By sequentially measuring changes in the absorbance or fluorescence of the reaction medium over time at said wavelengths, the concentration of a corresponding number of enzymes may be determined by formulating simultaneous equations of the first degree. See U.S. Pat. No. 3,925,162 (Kanno). See also, U.S. Pat. No. 3,718,433 entitled "Method of Analyzing of Ammonia, Urea, and Tyrozene", which also discloses methods of simultaneously performing biuret and allatonin tests on certain solutions.
For other papers and disclosures relating to spectrophotometric analysis of various blood serum components, please refer to West German Auslegeschrift 2558536 (Offenlegunstag, July 7, 1977); Luderer, "An Automated Method for the Enzymatic Determination of Triglycerides in Serum"; Banauch, et al., "A Glucose Dehydrogenase for the Determination of Glucose Concentrations in Body Fluids", Z. Klin, Chem. Klin, Biochem., 13.JG. 1975, S. 101-107; Kageyama, N., "A Direct Colorometric Determination of Uric Acid in Serum in Urin With Uricase-Catalase System", Clinica Chemica Acta, 31 (1971), 421-426. See also, U.S. Pat. Nos. 3,907,645 (Richmond); 3,703,591 (Bucolo et al.); 3,925,164 (Beaucamp, et al.); 4,102,646 (Sleeter). Also, please refer to E. I. Stern's, "The Practice of Absorption Spectro-Photometry", Wiley-Inter Science, New York, and U.S. Pat. No. 3,899,297 (Hirschfeld).
While considerable progress in the determination of blood serum components has been made, various practical considerations have somewhat limited the success of prior art methods. Ideally, simple, low cost, reagents or reagent sets exhibiting extended shelf life are needed to cover a wide range of serum components. Such reagents or reagent sets preferably should be suitable for use with samples maintained within normal temperature ranges to produce reaction media which are readily analyzed to provide statistically significant determinations. Often, due to the differing reaction kinetics of the component specific colorimetric reactions, analysis of multiple components in a single reaction medium may require numerous, sequential photometric determinations, first for one component, and then, substantially later for a second component. Preferably such methods should minimize the number of transfers for photometric analyses to which a single medium must be subjected, to thereby facilitate rapid and reliable processing of samples to be analyzed for multiple component content.